Comparative analysis of the active sites of orthologous endolysins of the Escherichia lytic bacteriophages T5, RB43, and RB49.

The methods of solution NMR, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study two zinc-containing L-alanyl-D-glutamate peptidases - endolysins of the pseudo T-even myoviruses RB43 and RB49 (EndoRB43 and EndoRB49, respectively), which are orthologous to the EndoT5, which is a zinc-containing L-alanyl-D-glutamate peptidase of the T5 siphovirus. The spatial conservation of the Zn2+-binding sites for the enzymes EndoT5, EndoRB43, and EndoRB49 was established, and the key role of Zn2+ ions in the stabilization of the spatial structures of these three peptidases was confirmed. We are showing here that the binding of the Zn2+ ion in the active center of EndoRB49 peptidase causes conformational rearrangements similar to those observed in the EndoT5 peptidase upon binding of Zn2+ and Ca2+ ions and lead to the formation of a catalytically active form of the enzyme. Therefore, the binding of the Zn2+ ion to the active site of EndoRB49 peptidase is a necessary and sufficient condition for functioning of this protein.

Pubmed ID: 33159938